Project Summary/Abstract A review of post-marketing surveillance reveals that humans receiving IL-1? inhibitors (e.g. Anakinra) have strikingly high (> 800 fold-increased risk) reporting of invasive group A Streptococcus (GAS) infections, including 11 fatal cases of necrotizing fasciitis. We found that Anakinra treatment, but not treatment a novel direct NLRP3 inflammasome inhibitor MCC950, increased mouse susceptibility to GAS. Surprisingly, GAS induced macrophage IL-1? production independently from canonical inflammasome regulatory components. Secreted GAS cysteine protease SpeB was necessary and sufficient for induction of non-canonical IL-1? signaling. Thus IL-1? acts a sensor to directly detect pathogen-associated proteolysis, an independent innate immune pathway running in tandem with the conventional inflammasome response, and which is blocked with Anakinra treatment. Based on this striking discovery, the goals of this program are three-fold. In Aim 1, we will understand how pharmacological modulation of IL-1? signaling, and consequent changes in macrophage and neutrophil innate immune and inflammatory function, can influence the pathogenesis and outcome of severe bacterial infections ? pneumonia and sepsis. Leading IL-1? receptor-blocking drug, Anakinra will be compared directly to an investigational small molecule NLRP3 inhibitor MCC950. We predict from our own clinical and experimental studies that higher IL-1? responses may be detrimental in lung infection but beneficial in systemic infection, and differ between pathogens inducing noncanonical IL-1? processing (GAS and Pseudomonas aeruginosa) compared a pathogen that only activates canonical NLRP3 inflammasome, methicillin-resistant Staphylococcus aureus (MRSA). In Aim 2, we provide data that IL-1? and IL-10 responses to MRSA predict risk of prolonged bacteremia and mortality, and that different contemporary antibiotic treatments influence MRSA virulence factor expression and the magnitude and pattern of cytokines including IL-1? and IL-10 produced by macrophages. Surveying all the common anti- MRSA drugs currently in clinical use, will provide a comprehensive analysis of how these ?indirect? effects of antibiotic treatment may influence disease pathogenesis and how immunomodulatory pharmacodynamics parameters identified in vitro and ex vivo with phagocytic cells translate to in vivo infection/treatment models. Finally, the caution that we raise with Anakinra from the FDA adverse event reporting has inspired us to carefully examine the full suite of anti-inflammatory therapies currently used in KD. Aim 3 provides a comprehensive pharmacodynamics analysis of the infectious risks associated with different KD therapeutics that may support the preferred use certain agents or guide adjunctive interventions such as prophylactic antibiotics to guarantee patient safety. The Nizet Lab is a leader in the study of microbial pathogenesis and innate immunity to inform novel approaches for treatment of leading bacterial pathogens; Dr. Sakoulas' clinical antibiotic pharmacology perspective complements studies of high translational significance.